Fructans are used as markers in kidney diagnostics and in particular to determine the glomerular filtration rate (GFR) as a test for kidney function.
Fructans are straight or branched chain oligosaccharides and polysaccharides with an sucrose terminal end. Fructans can have different physical properties, such as water solubility depending on the degree of branching and polymerization. Fructans occur in plants as carbohydrate reserves. As a natural product the fructans have an unpredictable length.
The fructans inulin and sinistrin are used in particular as markers in kidney function tests. Inulin and sinistrin are composed of 10 to 40 fructose units with a corresponding molecular weight of 1600 to 6500 Da. After intravenous injection, inulin and sinistrin are neither changed nor stored in the organism, but they are filtered out by the kidney glomeruli and are not reabsorbed again in the tubuli. The filtration of the fructans may vary according to their size.
In order to assess kidney function, it is usual to determine the time variation of the concentration of the marker in the blood after intravenous injection of said marker. To do so, blood samples have to be drawn. The concentration marker in the blood may, for example, be determined by enzymatic methods, as described in Kuehnle et al., Nephron, 62, 104-107 (1992). This method is time consuming, very cumbersome and of limited use.
A simpler alternative, based on fluorescein isothiocyanate labelled inulin (FITC-inulin) was described (M. Sohtell et al., Acta Physiol. Scand 119, 313-316 (1983); J. N. Lorenz and E. Gruenstein, Am. J. Physiol. (Renal Physiol. 45) 276, F172-F177 (1999). With this approach, also, blood is sampled and the fluorescein label of the FITC-Inulin is determined in the plasma. A very serious problem with this approach is that hemolysis, occurring during blood sampling, affects the determination of the concentration. Moreover, hemoglobin absorbs the excitation light at 480 nm very well, so less light is emitted and the apparent concentration is lower than in reality.
A disadvantage of inulin and FITC-inulin for the clinical routine analyses is their very low solubility in water. Hence the preparations containing insulin and FITC-insulin have to be heated to 90° C. until complete dissolution (Rieg, T. A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice. J. Vis. Exp. (75), e50330, doi:10.3791/50330 (2013)), as their aqueous solutions tend to crystallize during storage. Unfortunately, this causes a partial degradation of inulin to fructose. Furthermore, the solution has to be then dialysed for 24 hr at room temperature. This step is especially important to FITC-inulin in order to remove unconjugated FITC, but also the byproducts generated by the heating procedure. Dialysis substantially decreases the concentration of FITC-inulin. In addition, the low solubility of inulin and FITC-inulin makes it difficult to achieve a well defined concentration and to handle the marker during the injection.
Recently, a Cy5.5-inulin conjugate has been introduced by Perkin-Elmer (GFR-Vivo; application note by Peterson, J. D, Perkin-Elmer Corporation). An advantage of Cy5.5-inulin conjugate over FITC-inulin is related to the excitation/emission wavelengths of Cy5.5 (675/705 nm). The longer wavelength of Cy5.5 allows a deeper tissue penetration, but its use with a small animal imager requires the animals being anesthetized. Anesthesia, however, has an unpredictable impact on blood pressure (initial rise, decrease during the major phase of anesthesia, followed by a rise at the ending phase). Kidney perfusion and thus GFR are highly sensitive to blood pressure, with low blood pressure values resulting in low GFR values. Thus, a meaningful/reproducible GFR measurement is not possible under anesthesia.
A substantial improvement over the previous art was the introduction of a FITC-sinistrin conjugate as described in U.S. Pat. No. 6,995,019. This marker is much more water soluble than FITC-inulin and, in addition, no undesired circulatory reactions have been observed when using FITC-sinistrin. Furthermore its concentration change over time can be measured transcutaneously. There is, however, still the problem of the penetration depth into the skin at a given wave length (480 nm allow a depth of only a few mm) and thus the dosage of the marker has to be fairly high to obtain a clear (measurement) signal. Another problem of the transcutaneous measurement is skin pigmentation, as melanin in the skin absorbs light quite efficiently at 480 nm.
A major disadvantage of inulin and also sinistrin is that they are natural products; their composition is quite variable even within the same batch, and even more in different batches. For Regulatory Affairs this is not acceptable.